Lactobacillus acidophilus, as the main probiotic in the small intestine, has a role in modulating intestinal immune activity; however, its potential molecular regulatory mechanisms have rarely been investigated. To investigate the effects of Lactobacillus acidophilus LA85 in immunoprophylaxis, LA85 at the dose of 1 × 10⁸ CFU mL⁻¹, 1 × 10⁹ CFU mL⁻¹, and 1 × 10¹⁰ CFU mL⁻¹ were orally administered to immunocompromised ICR mice that had been treated intraperitoneally with 80 mg kg⁻¹ cyclophosphamide. The experimental results showed that LA85 could regulate the Notch signaling pathway and promote intestinal mucosal repair. And it could enhance the immune response of the body through the TLR4/NF-κB signaling pathway. 16S rDNA gene sequencing revealed that LA85 reduced the ratio of Firmicutes/Bacteroidetes and reshaped the gut microbiota. Furthermore, we discovered the correlation between Erysipelatoclostridium, Rikenella, and the intestinal Notch signaling pathway, and further discovered the potential mechanism of LA85 in intestinal mucosal repair. Based on the results of the study, we found that the effect of high doses of LA85 was more significant. Therefore, LA85 can be used as a dietary supplement to potentially enhance the immune capacity of patients.